Vol. 70 FORMATION OF e-(AMINOSUCCINYL)-LYSINE 373 4. Oa-(O-L-Aspartyl)-L-lysine and M-( -L-a8partyl)- Cummins, C. S. & Harris, H. (1956). J. gen. Microbiol. 14, L-lysine cyclize to X-(L-aminosuccinyl)-L-lysine in 583. 11 N-hydrochloric acid at 80°, but this derivative of Cummins,C. s. & Harris, H. (1958). J.gen.Microbiol.18,173. aminosuccinimide is much less stable than that Hausmann, W., Weisiger, J. R. & Craig, L. C. (1955). J. Amer. chem. Soc. 77, 723. formed from the corresponding e-aspartyl-lysines. Hirs, C. H. W., Moore, S. & Stein, W. H. (1954). J. Amer. 5. e-(Aminosuccinyl)-lysine is formed when chem. Soc. 76, 6063. bacitracin A is treated with 11 N-hydrochloric acid John, W. D. & Young, G. T. (1954). J. chem. Soc. p. 2870. at 800. It is also formed during acid hydrolysis of Lockhart, I. M. & Abraham, E. P. (1954a). Biochem. J. the cell walls of certain strains of lactobacilli. The 58, 633. cell walls of other strains of lactobacilli do not Lockhart, I. M. & Abraham, E. P. (1954b). Biochem. J. 58, yield this compound, although they contain lysine xlvii. and aspartic acid residues. Lockhart, I. M. & Abraham, E. P. (1956). Biochem. J. 62, 645. One of us (D.L.S.) is indebted to the Medical Research Mechanic, G. L. & Levy, M. (1957). Fed. Proc. 16, 220. Council for a scholarship. Infrared spectra were deter- Meloche, I. & Laidler, K. J. (1951). J. Amer. chem. Soc. 73, mined by Dr F. B. Strauss. The analysis was by Weiler and 1712. Strauss. Moore, S. & Stein, W. H. (1948). J. biol. Chem. 176, 367. REFERENCES Moore, S.& Stein,W.H. (1954). J. biol. Chem. 211, 893, 907. Newton, G. G. F. & Abraham, E. P. (1953). Biochem. J. 53, Abraham, E. P. (1957). Biochemistry of 8ome Peptide and 604. Steroid Antibiotics, p. 19. New York: John Wiley and Partridge, S. M. & Davis, H. F. (1950). Nature, Lond.,165,62. Sons, Inc. Peart, W. S. (1956). Biochem. J. 62, 520. Battersby, A. R. & Robinson, J. C. (1955). J. chem. Soc. Powell, J. F. & Strange, R. E. (1957). Biochem. 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Woiwod, A. J. (1949). J. gen. Microbiol. 3, 312. The Production of an Esterase Inhibitor from Schradan in the Fat Body of the Desert Locust BY M. L. FENWICK Department of Biochemi8try, Univer&ity of Leed8 (Received 28 March 1958) The oxidative conversion of the systemic insecti- pyridine nucleotide. O'Brien & Spencer (1953, cides schradan (bisdimethylaminophosphonous an- 1955) reported that many intact insect tissues hydride) and dimefox (bisdimethylaminofluoro- could produce the same schradan metabolite as did phosphine oxide) into powerful anticholinesterases liver, but all attempts to make an active tissue has been studied in mammalian liver homogenates homogenate were unsuccessful. Cockroach-gut (Davison, 1954, 1955; O'Brien, 1956, 1957; homogenates fortified with nicotinamide, diphos- Fenwick, Barron & Watson, 1957), and shown to be phopyridine nucleotide and Mg2+ ions failed to dependent on the presence of Mg2+ or Ca2+ ions, convert schradan (O'Brien, 1957) and dimefox nicotinamide and diphosphopyridine nucleotide. (Fenwick et al. 1957) into such compounds, and it The microsome fraction of liver cells could effect was suggested that a different mechanism might be the conversion if fortified with reduced diphospho- involved. 374 M. L. FENWICK I958 An investigation of the conversion of schradan directly proportional to enzyme concentration up to an by the tissues of the desert locust was undertaken optical-density change of 0-33. in order to determine whether there is a funda- Schradan conversion by intact ti88ue. Batches of fat-body mental difference of mechanism between mammals tissue (30 mg. wet wt.) were incubated in air for 30 min. at and insects. The choice ofinsect was governed by its 370 in 2 ml. of 'insect Ringer' solution (Thomsen, 1952), buffered with 10% (v/v) of -Oim-phosphate buffer, pH 7-4, size, availability and economic importance. in the presence of schradan at concentrations of 25, 50, 100 and 500/uM. The tissue was then homogenized in the medium, water was added to give a total of 1 ml./3-6 mg. MATERIALS AND METHODS of tissue, and the suspension was centrifuged for 2 mm. at Schradan, diphosphopyridine nucleotide (DPN), triphos- 600g. A volume (1 ml.) of supernatant was taken for phopyridine nucleotide (TPN) and flavine mononucleotide esterase measurement after removal of the upper layer of (FMN) were obtained from L. Light and Co. Ltd.; dimefox fat. Converting power was expressed as the mean of the from Fisons Pest Control Ltd.; flavinadenine dinucleotide four values oflog (100/b)/mg. dry wt./concn. of schradan (M) (FAD) from Sigma Chemical Co.; DPNH (reduced DPN) which were so obtained, where b is the percentage of the was prepared by the reduction of DPN with dithionite initial esterase which remained. (Colowick & Kaplan, 1955), and TPNEI (reduced TPN) by Dry weight and fat content were obtained by homo- the enzymic method of Nason & Evans (1953). p-Nitro- genizing a weighed sample of tissue in a small volume of phenyl acetate was synthesized by the method ofKaufmann water and shaking with ether. After a brief centrifuging, a (1909). portion of the ether extract was dried at room temperature Fourth-instar nymphs of the desert locust (Schistocerca and weighed. The aqueous suspension, freed of the ether gregaria Forsk.) were provided at regular intervals by the layer, was dried at 1100 and weighed. Anti-Locust Research Centre, 1 Princes Gate, London, Schradan conversion by homogenate8. A fat-body homo- S.W. 7. The insect cage stood in a thermoregulated room, genate in water was incubated for 3 hr. at 370 to reduce its and was lit and heated internally by an electric-light bulb Schradan-converting power. Schradan concentrations up which was automatically switched on at 8 a.m. and off at to mm had no significant effect on its esterase activity. midnight. The mean day temperature inside the cage was mm-Schradan was therefore usually used in subsequent 350, falling to 250 at night. A tray of water stood beneath conversion experiments, and schradan was omitted from the wire-gauze floor of the cage, and the humidity was controls. The components were placed in 6 in. x 1 in. further influenced by a jar of fresh or cold-stored green boiling tubes shaken in a water bath at 370 and open to the grass and a dish of drinking water in the cage. The grass air. A typical reaction mixture consisted of 0-2 ml. of fat- diet was supplemented by a mixture of grass meal, bran, body suspension, 0-2 ml. of inhibitor or activator dissolved dried whole milk and dried autolysed yeast (The Distillers in 0-25 m-sucrose and 0-1 ml. of 5 mM-schradan in 0-lm- Co. Ltd.) in the proportions 2: 2: 2: 1 (by vol.). phosphate buffer, pH 7-4. At the end of the incubation The tissues were removed from the decapitated insects period (usually 30 min.) water was added to stop the and placed in an ice-cooled dry beaker. Air sacs and reaction by dilution and cooling, and to give a convenient tracheae were generally not separated from the fat body concentration of esterase, which was then estimated. after it was found that their presence did not affect Centrifugal fractionation. A homogenate of 150-200 mg. schradan conversion. Testes were removed before the fat wet wt. of fat body/ml. of 0-25M-sucrose solution was body, as otherwise they tended to become incorporated in centrifuged for 2 min. in a bench centrifuge (about 600g), it. The unwashed tissue was homogenized with a glass and the supernatant was decanted after removal of the Potter-Elvehjem tube and pestle. Fat-body tissue is very upper layer of fat by suction. This did not reduce the soft, and five or six up-and-down movements of the pestle schradan-converting activity of the suspension. The super- were sufficient to release the fat from the cells. natant was then centrifuged at 00 for a further 30 min. at Estera8e determination. The principle of the method of 18000g. The resulting clear supernatant was carefully Huggins & Lapides (1947) was used, the rate of liberation pipetted off and replaced by an equal volume of sucrose ofp-nitrophenolfromp-nitrophenyl acetate being measured. solution in which the sedimented particles were resus- The substrate could be kept for up to a month at - 150 as pended. a 50 mM-solution in dry methanol. For each series of RESULTS estimations it was diluted (1/25) with water, and used within 1 hr. Esterase preparation (1 ml.) was added to Intact ti88U6 3 ml.